We propose to study the biochemical mechanisms that underlie the timing and integration of G protein-mediated signals in cells. G protein signaling modules control diverse cellular functions in response to equally diverse inputs. Consequently, G protein signaling is involved in many disease processes, and is the target of a huge number of drugs. G protein modules consist of a conserved group of interacting proteins: receptors, G protein Ga and Gbg subunits, GTPase activating proteins (GAP) and effector proteins. A mammalian cell typically expresses about 30 G protein-coupled receptors, half-dozen G proteins and GAPs and a dozen effectors. Mechanism of action is conserved, but varies quantitatively to allow different cells to respond to extracellular signals with a wide variety of kinetic patterns, intracellular outputs and modes of signal integration. Output from a G protein module quantitatively reflects a balance of receptor-catalyzed G protein activation and GAP-promoted deactivation. The rates of signal initiation and termination thus seem linked to the level of output, but cells can control response kinetics and response levels independently. We developed a quantitative framework for analyzing how receptors and GAPs interact to solve this problem. We will test mechanisms of receptor-GAP interaction and evaluate how and when each contributes to the temporal control of signaling. They include the ability of GAPs to stabilize the association of receptors and G proteins and to directly potentiate receptor function. We recently discovered that Gaq and Gbg, each of which stimulates phospholipase C-bs (PLC- bs) in response to different receptors, together stimulate the PLC-b3 isoform with strong synergism, 10 times the sum of the activities evoked by each subunit individually. Gaq-Gbg synergism is observed in diverse animal cells. We showed that Gaq-Gbg synergism can be explained by a classical two-state allosteric model, and we propose to test the physical basis of that interaction. Further, this model predicts that any two-state enzyme that is stimulated by two different ligands will display significant synergism only if its basal activity is very low, < 0.1% of maximum. We will test this prediction by evaluating constitutively active PLC-b3 mutants for loss of synergism and constitutively deactivated PLC- b2 mutants, which should acquire synergistic regulation. We will also use this prediction to search for novel regulatory enzymes that can act as coincidence detectors for two or more ligands.